1. Field of the Invention
The invention relates to peptides which comprise covalently linked T helper (Th) epitopes, cytoxic T lymphocyte (CTL) epitopes and epitopes which elicit a neutralizing antibody response (AbN) to an infectious agent, especially a parasitic or viral pathogen. Specific examples focus on application of the invention against Human Immunodeficiency Virus (HIV). The peptides have the further characteristic of evoking all three of these responses in hosts having a broad range of Major Histocompatibility Complex (MHC) types.
The invention is also directed to diagnostic methods for immune function in individuals infected with HIV which utilize the above-described peptides and is further directed to prophylactic or therapeutic vaccines which use the above-described peptides as a component of, or perhaps, as the sole active ingredient in the vaccine composition.
2. Description of the Related Art
Immune responses to HIV antigens elicited during natural infection may be a balance between those regulating viral infection and those antagonistic to the integrity of immune function (1-3). The determinants which weigh favorably or unfavorably upon this balance are not certain. The initial immune responsiveness of the host seems to influence the course of persistent HIV infection leading to progressive debilitating disease associated with increasing immunologic dysfunction (4-7). The virus may contain structures enabling it to evade the immune system, such as suppressive epitopes or masking carbohydrates, structures inducing clonal restriction (8-10), or structures that elicit deleterious effects such as antibodies which enhance viral infectivity (11-16) or autoreactive antibodies or T cells that contribute to the immunodeficiency (17-20).
The principal neutralizing determinant (PND) of the HIV envelope is located in the third hypervariable region or V3 loop between cysteine residues 301 and 331 (21-23). Antibodies to this region were initially demonstrated to be type specific in their neutralizing properties and more cross-reactive when examined by peptide binding ELISA (23-26), although more broadly neutralizing antibodies to the V3 loop have also been observed (27). Fortunately for synthetic vaccine development, such antibodies can be raised by immunization with short peptides (21,28,29). The protective efficacy of V3-specific antibodies to homologous cell-free virus challenge has been shown in chimpanzee challenge studies (15,30,31) and most recently protection against viral challenge was achieved by passive transfer into chimpanzees of a mouse-human IgG1 chimeric monoclonal antibody specific for the V3 loop (32). Sequence variation in the viral envelope protein in and outside (but affecting) this region results in both neutralization escape mutants, potential CTL escape mutants, and altered cellular tropism (33-37).
A high degree of genetic variability in HIV isolates can be found in infected individuals (38-40). HIV isolates from a given individual appear to change over the course of disease. Under immune pressure the virus appears to exhibit differences in phenotypic characteristics such as cytopathicity, replication rates, and cellular tropism during the course of infection. Evidence that the virus may be replicating continuously at low levels during infection and never achieve a state of "true latency" supports the view that HIV-1 produces a chronic active infection and selective mechanisms play an important role in viral persistence (33). Multiple distinct V3 regions encoding the PND of the envelope protein have been detected in isolates of HIV derived from peripheral blood mononuclear cells (PBMC), suggesting that positive selection leads to much diversity of HIV env genes in vivo (40). Nevertheless there is evidence that the PND contains conserved epitopes that are the targets of neutralizing antibodies generated by sequence divergent isolates and that a limited number of peptides from the PND can elicit neutralizing antibodies recognizing multiple isolates, albeit at lower titer, and probably lower affinity (24,25,41).
The criteria for an effective vaccine must be not only that it is safe, i.e., does not contain epitopes that elicit autoimmune or virus enhancing responses, but also that it is capable of eliciting both a cellular immune response and a neutralizing antibody response to all the potential HIV variants prevalent in the infected population. In addition, since the MHC molecule of a given individual will bind and recognize only a subset of potential antigenic determinants recognized by the species as a whole, a synthetic peptide vaccine must also incorporate enough antigenic determinants to elicit recognition by T cells of most HLA types.
Accordingly, in a previous study we constructed six synthetic peptides of 20-33 residues each that correspond to six multideterminant T helper regions of the HIV envelope (42). Called cluster peptides, these span clusters of distinct but overlapping T helper epitopes recognized by proliferating T cells of three or four haplotypes of mice. These cluster peptides were tested for their ability to stimulate T cell responses in mice immunized with recombinant gp 160 (rgp160) and in peripheral blood lymphocytes of humans infected with HIV. Mice were also immunized with the cluster peptides to test for the induction of T cells responding to intact gp160 in vitro. Cluster peptides 3, 4, and 6 (see sequences in Table I) stimulated T cells from mice of all four MHC haplotypes immunized with rgp160; and when mice were immunized with the cluster peptide, elicited T cell responses capable of recognizing the whole envelope protein in vitro. Cluster peptide 1, also used in this current study, stimulated proliferation strongly in only one strain of mice, despite the fact that the three other strains recognized components of the multideterminant region from which this peptide was made. Thus, the whole had less activity than the sum of its parts (42). Cluster peptides 1, 3, 4, and 6 stimulated significant IL-2 responses in peripheral blood lymphocytes of HIV-positive, influenza positive humans in 64, 73, 52, and 58% of tested cases respectively. It is of interest to note that these high responses were observed despite the fact that the subjects tested were presumably infected with a large number of different substrains of HIV. Cluster peptides 1, 3, and 4 have sequences relatively conserved among North American and European isolates of HIV, and cluster peptide 6 spans the boundary between conserved and variable sequences (43).
A successful peptide vaccine should be capable of eliciting T helper (Th) and cytotoxic T lymphocytes (CTL) responses as well as a neutralizing antibody response in vaccinees of multiple HLA types. Major histocompatibility complex (MHC) class I-restricted CTL appear to play a central role in the recovery from viral infection (81). Although exogenous lymphokines can substitute for T-cell help in the maturation of CTL precursors in vitro, the role of Th in priming CTL in vivo still remains poorly understood, compared to Th-B-cell collaboration. Although much evidence for a helper requirement in CTL induction exists (82-90), there is also evidence for CTL responses independent of help (85,91-95). Further, no study to date has shown a necessity for help requiring covalent linkage of a helper antigenic determiant to a CTL determinant, analogous to the linkage of carrier to hapten in cognate help for B cells. This lack of evidence may be due to the fact that the targets of CTL are whole cells, and immunization until recently required whole cells (or tissue grafts) or live viruses. The closest one could come to suggesting determinant linkage was to show that the helper determinant and CTL determinant had to be on the same skin graft to induce rejection (89), but this could not be explored further at the molecular level. Now that the possibility of peptide immunization for CTL induction has been demonstrated (96-100), it becomes feasible to address this question using peptides comprising both helper and CTL determinants. Although recent evidence indicated that a helper site is beneficial (90,101,102), it was not clear if the helper and CTL sites needed to be linked. Indeed, uncoupled helper and CTL epitope peptides were effective in two studies (90,102) and not tested in the other (101), but in the former studies, the mixture of helper and CTL determinant peptides was administered in incomplete Freund's adjuvant emulsion, which sequesters the two peptides in the same microenvironment, or was given at high dose for multiple immunizations.